This research addresses the general topic of organization and regulation of tRNA genes in Escherichia coli. The specific tRNAs that are being studied in this laboratory are the members of the threonine family, which are encoded by four genes. One is part of a ribosomal RNA operon, where it is sandwiched between two 5S rRNA genes in the distal region. Two others belong to a mixed-function operon, one coding for a protein (elongation factor EF-Tu) in addition to four tRNA species. These three genes are well characterized; each and its neighbors has been completely sequenced. The fourth gene, whose existence has only recently been suspected, will be the subject of this proposal. We would like to know exactly where the gene is located, whether it is a duplicate of one of the others or not, and whether it is clustered with other tRNA genes or part of a mixed-function operon. The specific goals for this grant are the following: (1) To identify by hybridization methods a restriction fragment that carries it. Several different end-labeled or nick-translated probes will be used, including bulk tRNA, threonine acceptor tRNA, partially purified tRNA-4-Thr, and phage DNA carrying homologous genes. (2) To clone the gene in a plasmid vector. Chromosomal DNA fragments of the appropriate size will be ligated into pBR322 or pUC18, and transformants will be screened with one of the above probes. (3) To map the gene physically within the cloned fragment. A restriction map will be constructed and the gene location will be identified by hybridization. (4) To determine if other tRNA genes might also be included in the fragment. Southern hybridization will be done with a bulk tRNA probe. (5) To use this recombinant plasmid to map the gene. The recombinant plasmid will be integrated into the chromosome at the region of homology, and the plasmid antibiotic-resistance gene will be used as a marker in standard genetic mapping experiments.